Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development Haikel N. Bogalea, Tales V. Pascinib, Sachie Kanatanic, Juliana M. Sáb, Thomas E. Wellemsb,1, Photini Sinnisc, Joel Vega-Rodríguezb, and David Serrea,d,1 aInstitute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201; bLaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; cDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; and dDepartment of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201 Contributed by Thomas E. Wellems, January 18, 2021 (sent for review November 11, 2020; reviewed by Daniel E. Neafsey and Ashley M. Vaughan) Despite the critical role of Plasmodium sporozoites in malaria transmis- are ejected with the mosquito saliva into the dermis during the sion, we still know little about the mechanisms underlying their de- probing phase of a bite (16, 17). Once in the skin, sporozoites velopment in mosquitoes. Here, we use single-cell RNA sequencing to move rapidly to locate blood vessels and enter the blood circula- characterize the gene expression profiles of 16,038 Plasmodium ber- tion, which carries them to the liver, where they enter hepatocytes ghei sporozoites isolated throughout their development from midgut and develop into liver stages. During their migration, from the oocysts to salivary glands, and from forced salivation experiments. Our mosquito midgut to the mammalian liver, sporozoites do not show results reveal a succession of tightly regulated changes in gene expres- major morphological changes but go through important develop- sion occurring during the maturation of sporozoites and highlight can- mental changes. For example, sporozoites collected from oocysts didate genes that could play important roles in oocyst egress, or from the hemolymph can cause successful mammalian infec- sporozoite motility, and the mechanisms underlying the invasion of tions but these sporozoites are, overall, much less infectious than mosquito salivary glands and mammalian hepatocytes. In addition, the salivary gland sporozoites (18–20). Conversely, sporozoites col- single-cell data reveal extensive transcriptional heterogeneity among lected from a salivary gland and injected into the hemolymph parasites isolated from the same anatomical site, suggesting that Plas- show reduced infectivity for the mosquito salivary glands (21). MICROBIOLOGY modium development in mosquitoes is asynchronous and regulated These changes in infectivity are mirrored by changes in mRNA by intrinsic as well as environmental factors. Finally, our analyses show expression and protein abundance that have been characterized a decrease in transcriptional activity preceding the translational repres- for rodent parasites and, to a lesser extent, for Plasmodium falci- sion observed in mature sporozoites and associated with their quies- parum (22, 23). Combined with elegant reverse genetic experi- cent state in salivary glands, followed by a rapid reactivation of the ments, these analyses have highlighted some of the molecular transcriptional machinery immediately upon salivation. processes underlying sporozoite maturation, and revealed key Plasmodium genes involved in sporozoite egress from the oocyst malaria | single-cell analysis | gene expression [e.g., PbSERA5 (24), SIAP-1 (25), and PCRMPs (26)], and in the alaria is a disease caused by unicellular parasites of the Significance MPlasmodium genus that are transmitted to humans by the bites of infected female Anopheles mosquitoes. Socioeconomic A better understanding of the molecular mechanisms under- development (1–4), combined with the use of extensive entomo- lying the development of Plasmodium parasites in Anopheles logical controls (5, 6) and improved antimalarial drug develop- mosquitoes would facilitate the development of malaria vac- ment and distribution (7, 8), have significantly decreased the cines and of novel strategies to interrupt disease transmission. mortality associated with malaria over the last 50 y. However, the We characterized, at the single-cell level, the gene expression disease remains a heavy burden affecting more than 200 million profiles of sporozoites from the rodent malaria parasite Plas- people and responsible for half a million deaths annually (9). An modium berghei, throughout their development in mosquitoes efficient malaria vaccine remains elusive, but encouraging progress and upon salivation. Our analyses reveal heterogeneity in gene has been achieved in recent years with the development and testing expression among parasites isolated from the same anatomical of vaccines using recombinant proteins (10, 11) and attenuated location, suggesting that parasite development is asynchro- parasites (12–14) from the human infective stage, the sporozoite. nous in mosquitoes. In addition, our results highlight the role However, despite renewed interest in Plasmodium mosquito stages of gene expression changes in regulating the ability of sporo- for vaccine development, many of the molecular processes regu- zoites to remain quiescent in the salivary glands, and their lating sporozoite development remain unclear. rapid reactivation upon salivation. Following the ingestion of male and female gametocytes during a blood feeding, fertilization occurs in the mosquito midgut, pro- Author contributions: J.M.S., T.E.W., P.S., J.V.-R., and D.S. designed research; H.N.B., T.V.P., S.K., and J.M.S. performed research; T.E.W., P.S., and J.V.-R. contributed new re- ducing zygotes that undergo meiosis and develop into motile agents/analytic tools; H.N.B., P.S., J.V.-R., and D.S. analyzed data; and H.N.B., T.E.W., P.S., ookinetes. The ookinete traverses the midgut epithelium and ma- J.V.-R., and D.S. wrote the paper. tures into an oocyst. On the basal surface of the midgut epithelium, Reviewers: D.E.N., Harvard University; and A.M.V., Seattle Children’s Research Institute. the sessile oocyst undergoes multiple cycles of DNA replication The authors declare no competing interest. and cell divisions, which leads to the production of thousands of ’ This open access article is distributed under Creative Commons Attribution-NonCommercial- sporozoites that are released into the mosquito s hemolymph. The NoDerivatives License 4.0 (CC BY-NC-ND). crescent-shaped sporozoites are then transported by the hemo- 1To whom correspondence may be addressed. Email: [email protected] or dserre@ lymph and ∼20% of them successfully enter the salivary glands, som.umaryland.edu. where they wait to be inoculated into a mammalian host, remaining This article contains supporting information online at https://www.pnas.org/lookup/suppl/ viable for days to weeks (15). Transmission to the mammalian host doi:10.1073/pnas.2023438118/-/DCSupplemental. occurs when a small number of sporozoites, typically less than 100, Published March 02, 2021. PNAS 2021 Vol. 118 No. 10 e2023438118 https://doi.org/10.1073/pnas.2023438118 | 1of9 Downloaded by guest on September 25, 2021 recognition and invasion of the mosquito salivary glands [e.g., 128 to 248 million paired-end reads of 75 base pairs (SI Appendix, TREP (27), MAEBL (28), and CSP (29)]. However, despite these Table S1). Between 0.6 and 67.2% of the reads mapped to the P. important studies, several outstanding questions remain regarding berghei ANKA genome (38), providing, on average, 20.2 million the regulation and development of Plasmodium sporozoites in Plasmodium reads per sample. Most of the remaining reads mosquitoes. For example, it is not clear whether the maturation of mapped to the An. stephensi genome and represented contami- sporozoites is primarily driven by extrinsic (e.g., the location of the nation by mosquito RNA. After stringent quality filters (Materials sporozoites in the mosquito) or intrinsic factors (e.g., the age of and Methods), we obtained between 82 and 4,630 single Plasmo- the sporozoites) as these parameters are confounded in most dium cell transcriptomes from each library, for a total of 16,038 studies. Similarly, we do not know whether all sporozoites at the individual sporozoite transcriptomes, each characterized by an same anatomical location are identically regulated or whether they average of 1,033 unique reads per parasite (235 to 5,867) (SI mature asynchronously. These questions have been difficult to Appendix,TableS1). rigorously address due to technical limitations of available meth- ods. In order to have sufficient amounts of material for analysis, scRNA-Seq Reveals Heterogeneous and Overlapping Changes in Gene genomic and proteomic studies typically rely on bulk approaches Expression during P. berghei Sporozoite Development. We charac- utilizing large numbers of parasites, which only provide “averages” terized the transcriptomes of 16,038 sporozoites: 614 sporozoites on these populations and mask potential heterogeneity among collected from disrupted oocysts, 2,147 sporozoites isolated from individual parasites. The advent of single-cell technologies allows the mosquito hemolymph, 5,979 sporozoites dissected from mos- us to examine the regulation of individual cells and has already quitoes’ salivary glands, and 7,298 sporozoites obtained after forced provided exciting new insights on the biology of Plasmodium salivation experiments (SI Appendix,TableS1). After excluding